Methods for preventing and treating obesity in patients with mc4 receptor mutations

ABSTRACT

Methods are provided for treating health conditions associated with altered MC4 receptor activity with melanin concentrating hormone receptor antagonists. Such compounds may be used, for example, to treat or prevent obesity and/or overeating, and to reduce body mass index, in patients carrying one or more MC4 receptor mutations.

FIELD OF THE INVENTION

This invention relates generally to methods for treating healthconditions associated with altered MC4 receptor activity, and morespecifically to the use of melanin concentrating hormone receptorantagonists for the prevention and treatment of obesity and overeatingin patients carrying MC4 receptor mutations.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 Human MC4R amino acid sequence (Gantz et al. (1993) J. Biol.Chem. 268:15174-79)

BACKGROUND OF THE INVENTION

Obesity is the most common nutritional problem in developed countries.By some estimates, obesity affects more than half of the population ofthe United States, where about 300,000 deaths annually are attributableto this condition. Obesity often leads to serious health conditions,such as diabetes, atherosclerosis, pulmonary embolism, coronary arterydisease, hypertension, stroke, diabetes, sleep apnea, deep-veinthrombosis, hyperlipidemia and some cancers, and complicates numerouschronic conditions such as respiratory diseases, osteoarthritis,osteoporosis, gall bladder disease and dyslipidemias. Fortunately,however, many of the conditions caused or exacerbated by obesity can beresolved or dramatically improved by weight loss.

Once considered merely a behavioral problem (i.e., the result ofvoluntary hyperphagia), obesity is now recognized as a complexmultifactorial disease involving defective regulation of food intake,food-induced energy expenditure and the balance between lipid and leanbody anabolism. Both environmental and genetic factors play a role inthe development of obesity. As a result, treatment programs that focusentirely on behavior modification have limited efficacy and areassociated with recidivism rates exceeding 95%. Pharmacotherapy is nowseen as a critical component of weight loss and subsequent weightmanagement.

The central melanocortin system is critical for the regulation of foodintake and energy balance. Within this system, melanocortins (a varietyof different peptide products resulting from post-translationalprocessing of pro-opiomelanocortin) stimulate or inhibit food intake viaaction at one or more melanocortin receptors. Alterations inmelanocortin receptor activity have been shown to affect food intake.

Five melanocortin receptor subtypes have been described to date. Ofthese, melanocortin 4 receptor (MC4R) is the most abundant and mostwidely distributed in the brain. MC4R plays a specific role in appetiteregulation. In both humans and mice, interruption of signaling at MC4Rresults in overeating, increased body-mass index and obesity. Inaddition, a variety of mutations in MC4R have been shown to cause morbidobesity in humans. Most known genetic mutations that result in obesityare recessive and cause only rare forms of obesity that occur incombination with endocrine abnormalities. Mutations in MC4R, however,can be dominant and are the most frequent known cause of severe obesity,estimated to occur in 3-5% of obese patients. MC4R is a 332-amino acidprotein that belongs to the family of seven transmembrane Gprotein-coupled receptors (GPCR) and signals via adenylate cyclase. Thisreceptor is expressed primarily throughout the brain, and is activatedby a melanocortin peptide known as alpha-melanocyte stimulating hormone(alphaMSH). MC4R agonists such as alphaMSH have been shown to reducefood intake (i.e., they produce an anorexigenic effect), whileantagonists of this receptor stimulate food intake (i.e., they producean orexigenic effect).

Unfortunately, peptides such as alphaMSH are typically broken down bythe digestive system, so that peptides are not usually suitable for oraladministration to patients. In this regard, so-called small moleculepharmaceutical agents often have the advantage of being suitable fororal administration.

Other signaling pathways also contribute to obesity. Melaninconcentrating hormone, or MCH, is a cyclic 19 amino acid hypothalamicpeptide that functions as a regulator of food intake and energy balance,serving as a neurotransmitter in the lateral and posterior hypothalamus.MCH mRNA is overexpressed in ob/ob C57BL/6J mice, and mice with atargeted deletion of the MCH gene are characterized by reduced bodyweight, due to decreased feeding and increased metabolic rate. ICVadministration (i.e., injection directly into the ventricles of thebrain) of MCH has been shown to produce a mild orexigenic effect inrodents.

MCH activity is mediated via binding to specific receptors, of which MCHtype 1 (MCHR1) and type 2 (MCHR2) receptors have been identified. MCHR1is a 353 amino acid, 7-transmembrane, alpha-helical, G-coupled proteinreceptor, initially reported by Kolakowski et al. (1996) FEBS Lett.398:253-58; Lakaye et al. (1998) Biochim. Biophys. Acta 1401:216-220;Chambers et al. (1999) Nature 400:261-65; and Saito et al. (1999) Nature400:265-69. Upon binding MCH, MCHR1 receptors expressed in HEK 293 cellmediate a dose dependent release of intracellular calcium. Cellsexpressing MCH receptors have also been shown to exhibit a pertussistoxin sensitive dose-dependent inhibition of forskolin-elevated cyclicAMP, indicating that the receptor couples to a G_(i/o) G-protein alphasubunit. MCHR2 (An et al. (2001) Proc. Natl. Acad. Sci. USA98:7576-7581; Sailer et al. (2001) Proc. Natl. Acad. Sci. USA98:7564-7569; Hill et al. (2001) J. Biol. Chem. 276:20125-20129; Mori etal. (2001) Biochem. Biophys. Res. Commun. 283:1013-1018) has an overallamino acid identity of more than 30% with MCHR1, and is detected in mostregions of the brain, with an expression pattern similar to that ofMCHR1.

Although dysfunctions of various neurotransmitter and hormonal signalingpathways are known to contribute to obesity, the interrelationshipsamong these pathways are poorly understood. For example, it has not beenknown which pathways are upstream and which downstream of the MC4system. In this regard, modulating the activity of a pathway upstream ofMC4R would not be expected to correct the phenotype resulting from agenetic defect in MC4R, while modulating the activity of a pathwaydownstream of MC4R might be expected to have some impact. Additionally,such impact would be of unpredictable and indeterminate magnitude, atleast in the absence of any experimental data from which to extrapolate.

As a result, attempts to identify agents that decrease food intake inpatients with obesity-promoting MC4R mutations have focused on theidentification of agents that specifically affect the expression oractivity of MC4R. To date, however, no such agents have been developedand marketed for medical use. The current limited understanding of themolecular and genetic factors contributing to the development of obesityhas hampered the search for effective agents capable of inhibiting foodintake in individuals with diminished MC4 receptor activity.

Accordingly, there is a need in the art for methods, especially methodsemploying small molecule non-peptide agents, that are capable ofinhibiting food intake in obese individuals carrying MC4 receptor genemutations that are associated with diminished MC4 receptor activity. Thepresent invention fulfills this need, and provides further relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful for thetreatment of overeating or obesity in patients carrying an MC4Rmutation. Compositions generally comprise an effective amount of one ormore MCH receptor antagonists, in combination with a physiologicallyacceptable carrier or excipient.

Within certain aspects, the present invention provides methods fortreating obesity in a mammalian patient. Such methods comprisedetermining whether or not the obese patient carries a melanocortin 4receptor (MC4R) mutation that is associated with obesity and, if thepatient carries such a mutation, administering an amount of a non-toxicmelanin concentrating hormone (MCH) receptor antagonist effective toreduce either or both of (1) food consumption and/or (2) body mass indexin the patient upon sustained administration.

Within further aspects, methods are provided for preventing arecrudescence of obesity in a mammalian patient. Such methods comprisedetermining whether or not the previously obese patient carries amelanocortin 4 receptor (MC4R) mutation that is associated with obesityand, if the patient carries such a mutation, administering to thepatient an amount of a non-toxic melanin concentrating hormone (MCH)receptor antagonist effective to reduce either or both of (1) foodconsumption and/or (2) body mass index in the patient upon sustainedadministration.

Methods are further provided for preventing obesity in a mammalianpatient. Such methods comprise determining whether or not the patientcarries a melanocortin 4 receptor (MC4R) mutation that is associatedwith obesity and, if the patient carries such a mutation, administeringto the patient an amount of a non-toxic melanin concentrating hormone(MCH) receptor antagonist effective to reduce either or both of (1) foodconsumption and/or (2) body mass index in the patient upon sustainedadministration.

Within further aspects, the present invention provides methods fortreating or preventing obesity (e.g., preventing a recrudescence ofobesity) in a patient with an MC4R mutation, comprising administeringsuch an effective amount of a non-toxic MCH receptor antagonist to apatient previously determined to carry such a mutation.

These and other aspects of the present invention will become apparentupon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph illustrating the effect of an MCH receptor antagoniston food consumption in rats with reduced MC4 receptor activity. Theunshaded bar indicates the amount of food, in grams, consumed in a twohour period by rats treated with vehicle (saline) alone. The lightlyshaded bar indicates the amount of food consumed in a two hour period byrats treated with 6 nmol HSO14 (an MC4 receptor antagonist),administered by direct injection to the lateral ventricle. The dark barindicates the amount of food consumed in a two hour period by ratstreated with 20 mg/kg MCH receptor antagonist orally 30 minutes beforeICV administration of HSO14.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides methods for use inpreventing or treating overeating and/or obesity in patients with one ormore MC4R mutations. Compositions useful in the methods provided hereingenerally comprise a non-toxic MCH receptor antagonist. Suchcompositions may be administered to a patient with an MC4R mutation, forexample, to reduce food intake, BMI and/or obesity.

Terminology

A “patient” is any individual being considered for treatment with an MCHreceptor antagonist. Patients include humans, as well as other mammalssuch as companion animals and livestock, and are either obese or are atrisk for a recrudescence of obesity.

A “melanocortin 4 receptor (MC4R) gene” is a naturally-occurringnucleotide sequence that encodes MC4R (i.e., a G-protein coupledreceptor that is activated by alphaMSH and comprises an amino acidsequence that is at least 90% identical to SEQ ID NO:1) or would soencode MC4R (as determined by the precise chromosomal location,association with specific flanking sequences, comparison with allelicsequences, or like indications of gene locus identity) but for thepresence of one or more nonsense, missense, frameshift, insertion ordeletion mutations. The determination as to whether a nucleotidesequence is at least 90% identical to SEQ ID NO:1 is made using only theportions of SEQ ID NO:1 that align with the predicted protein product ofthe patient's MC4R gene. In other words, the protein product predictedfor the gene if all frameshifted coding regions (if any) were in frameand all inserted or deleted regions (if any) were not figured in to thecalculation. Such a determination is made using, for example, a ClustalWalignment. The term “MC4R gene” encompasses both the coding region andany introns or upstream or downstream regions that are tightly linked tothe MC4R locus.

A “melanocoltin 4 receptor (MC4R) gene” is a naturally-occurringnucleotide sequence that encodes an MC4R (i.e., a G-protein coupledreceptor that comprises an amino acid sequence that is at least 90%identical to SEQ ID NO:1). The encoded MC4R sequence may be truncatedrelative to SEQ ID NO:1; in such cases, the percent identity isdetermined using only the portion of SEQ ID NO:1 that aligns with theMC4R encoded by the patients MC4R gene using, for example, a ClustalWalignment. The term “MC4R gene” encompasses both the coding region andany introns or upstream or downstream regions that are tightly linked tothe MC4R locus.

Patients are said to “carry at least one MC4R mutation” if thenucleotide sequence of one or both of the patient's MC4R genes containsat least one sequence feature that results in a decrease in receptorfunction or is otherwise determined to be associated with obesity. AnMC4R mutation may be located in an upstream region, coding region,intron or downstream region of an MC4R gene. An MC4R mutation isgenerally a sequence alteration (e.g., any nucleotide deletion,insertion, or substitution) or other modification (e.g., an alteredmethylation state) relative to a reference MC4R sequence for a non-obesemember of the patient's species. An appropriate reference sequence forhumans is the MC4R sequence available at GenBank Accession NumberL08603, a translation of which is provided herein as SEQ ID NO:1, andappropriate reference sequences for other animals may be obtained usingconventional molecular biological techniques, using the human sequenceas a probe. A determination as to whether a patient carries at least oneMC4R mutation may be performed using standard techniques, such as PCR orRFLP mapping, with or without isolation of the MC4R gene. If priorgenetic testing has been done, such a determination may be convenientlymade by review of the patient's medical chart.

A mutation is considered to be “associated with obesity” if the mutationis identified in one or more obese patients, and is present at asignificantly lower frequency in a non-obese population (as determinedby any standard parametric test of statistical significance). MC4Rmutations currently known to be associated with obesity include, but arenot limited to, frameshift mutations (e.g., deletion of CTCT at codon211, resulting in a truncated protein, or insertion of four nucleotidesat codon 244), nonsense mutations (e.g., at codon 35, resulting in atruncated protein), and missense mutations (e.g., resulting in aminoacid substitution(s) at position 11, 18, 30, 37, 50, 58, 78, 98, 102,103, 112, 137, 150, 165, 170, 250, 252, 274, 301 and/or 317). Thepresent invention encompasses treatment of patients with any MC4Rmutation(s) that are currently known or are subsequently determined tobe associated with obesity.

As used herein, a patient is considered “obese” if the patient's bodymass index is greater than 28. Body mass index (BMI) may be readilycalculated using the following formula:BMI=(weight in kg)/(Height in meters)²

The term “MCH receptor” refers to a naturally-occurring mammalian (e.g.,human, dog, cat, or monkey) MCH type 1 or type 2 receptor such as theMCH type 1 receptor (MCHR1; e.g., Lakaye et al., supra) and the MCH type2 receptor (MCHR2; An et al., supra; Sailer et al., supra; Hill et al.,supra; Mori et al., supra). SEQ ID NOs:1 and 2 of WO 03/060475 recitethe DNA and amino acid sequences, respectively, of a Cynomolgus macaqueMCH1R.

A “MCH receptor antagonist” is a compound that detectably inhibits MCHbinding to one or more MCH receptors and/or inhibits MCHreceptor-mediated signal transduction, as measured using therepresentative assays provided in Examples 1 and 2 herein. Antagonistsfor use within the context of the present invention are generallynon-toxic. Within certain embodiments, an MCH receptor antagonist has arelatively low molecular weight (e.g., less than 700 amu) and ismulti-aryl (i.e., has a plurality of unfused or fused aryl groups),non-peptide and amino acid free. Such compounds include, but are notlimited to, substituted analogues of benzimidazole,1-benzyl-4-aryl-piperazine, 1-benzyl-4-aryl-piperidine, andphenylcycloalkylmethylamino and phenylalkenylamino compounds. Anantagonist binds “specifically” to MCH receptor if it binds to an MCHreceptor (total binding minus nonspecific binding) with a K_(i) that is10-fold, preferably 100-fold, and more preferably 1000-fold, less thanthe K_(i) measured for MCH receptor antagonist binding to other Gprotein-coupled receptors. An antagonist binds with “high affinity” ifthe K_(i) at an MCH receptor is less than 1 micromolar, preferably lessthan 500 nanomolar, 100 nanomolar or 10 nanomolar. MCH receptorantagonists preferably have minimal agonist activity (i.e., induce anincrease in the basal activity of the MCH receptor that is less than 5%of the increase that would be induced by one EC₅₀ of MCH), and morepreferably have no detectable agonist activity within the assaydescribed in Example 3).

The term “nontoxic” as used herein shall be understood in a relativesense and is intended to refer to any substance that has been approvedby the United States Food and Drug Administration (“FDA”) foradministration to mammals (preferably humans) or, in keeping withestablished criteria, is susceptible to approval by the FDA foradministration to mammals (preferably humans). In addition, a highlypreferred nontoxic compound generally satisfies one or more of thefollowing criteria: (1) does not substantially inhibit cellular ATPproduction; (2) does not significantly prolong heart QT intervals; (3)does not cause substantial liver enlargement, and (4) does not causesubstantial release of liver enzymes.

As used herein, a compound that “does not substantially inhibit cellularATP production” is a compound that satisfies the criteria set forth inExample 4, herein. In other words, cells treated as described in Example4 with 100 μM of such a compound exhibit ATP levels that are at least50% of the ATP levels detected in untreated cells. In more highlypreferred embodiments, such cells exhibit ATP levels that are at least80% of the ATP levels detected in untreated cells.

A compound that “does not significantly prolong heart QT intervals” is acompound that does not result in a statistically significantprolongation of heart QT intervals (as determined byelectrocardiography) in guinea pigs, minipigs or dogs uponadministration of twice the minimum dose yielding a therapeuticallyeffective in vivo concentration. In certain preferred embodiments, adose of 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administeredparenterally or orally does not result in a statistically significantprolongation of heart QT intervals. By “statistically significant” ismeant results varying from control at the p<0.1 level or more preferablyat the p<0.05 level of significance as measured using a standardparametric assay of statistical significance such as a student's T test.

A compound “does not cause substantial liver enlargement” if dailytreatment of laboratory rodents (e.g., mice or rats) for 5-10 days withtwice the minimum dose that yields a therapeutically effective in vivoconcentration results in an increase in liver to body weight ratio thatis no more than 100% over matched controls. In more highly preferredembodiments, such doses do not cause liver enlargement of more than 75%or 50% over matched controls. If non-rodent mammals (e.g., dogs) areused, such doses should not result in an increase of liver to bodyweight ratio of more than 50%, preferably not more than 25%, and morepreferably not more than 10% over matched untreated controls. Preferreddoses within such assays include 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or50 mg/kg administered parenterally or orally.

Similarly, a compound “does not promote substantial release of liverenzymes” if administration of twice the minimum dose yielding atherapeutically effective in vivo concentration does not elevate serumlevels of ALT, LDH or AST in laboratory rodents by more than 100% overmatched mock-treated controls. In more highly preferred embodiments,such doses do not elevate such serum levels by more than 75% or 50% overmatched controls. Alternately, a compound “does not promote substantialrelease of liver enzymes” if, in an in vitro hepatocyte assay,concentrations (in culture media or other such solutions that arecontacted and incubated with hepatocytes in vitro) equivalent totwo-fold the minimum in vivo therapeutic concentration of the compounddo not cause detectable release of any of such liver enzymes intoculture medium above baseline levels seen in media from matchedmock-treated control cells. In more highly preferred embodiments, thereis no detectable release of any of such liver enzymes into culturemedium above baseline levels when such compound concentrations arefive-fold, and preferably ten-fold the minimum in vivo therapeuticconcentration of the compound.

A “prodrug” is a compound that may not be an MCH receptor antagonist,but is modified in vivo, following administration to a patient, toproduce such an antagonist. For example, a prodrug may be an acylatedderivative of an MCH receptor antagonist. Prodrugs include compoundswherein hydroxy, amine or sulfhydryl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino or sulfhydryl group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups within an MCHreceptor antagonist.

Melanin Concentrating Hormone Receptor Antagonists

As noted above, the present invention provides compositions and methodsuseful for the treatment of overeating and obesity, and for reducingbody mass index, in patients carrying an MC4R mutation. Compositionsprovided herein generally comprise a non-toxic melanin concentratinghormone (MCH) receptor antagonist. Such antagonists may be specific fora particular MCH receptor (e.g., type 1 or type 2), or may function atmultiple MCH receptors. MCH receptor antagonists for use within thecompositions provided herein are, within certain embodiments, lowmolecular weight (e.g., less than 700 amu), multi-aryl, non-peptide andamino acid free.

As noted above, MCH receptor antagonists for use herein detectablyinhibit MCH binding to MCHR1 and/or MCHR2 receptor (as determined usinga standard in vitro MCH receptor ligand binding assay and/or calciummobilization assay) at submicromolar concentrations, preferably atnanomolar concentrations, and more preferably at subnanomolarconcentrations. References herein to a “MCH receptor ligand bindingassay” refer to the standard in vitro receptor binding assay provided inExample 2. Briefly, a competition assay may be performed in which an MCHreceptor preparation is incubated with labeled (e.g., ¹²⁵I) MCH andunlabeled test compound. Within the assays provided herein, the MCHreceptor used is preferably a mammalian MCHR1 or MCHR2 receptor, morepreferably a human or monkey MCHR1 or MCHR2 receptor. The MCH receptorpreparation may be, for example, a membrane preparation from HEK293cells that recombinantly express a human MCH receptor (e.g., GenbankAccession No. Z86090), monkey MCHR1 receptor (such as the MCHR1 sequenceprovided in SEQ ID NO:1 of WO 03/060475), or human MCHR1/humanbeta-2-adrenergic chimeric receptor.

Incubation with an MCH receptor antagonist results in a decrease in theamount of label bound to the MCH receptor preparation, relative to theamount of label bound in the absence of the antagonist. Preferably, anMCH receptor antagonist exhibits a K_(i) at an MCH receptor of less than1 micromolar, binding specifically and with high affinity to an MCHreceptor. More preferably, such a compound exhibits a K_(i) at an MCHreceptor of less than 500 nM, 100 nM, 20 nM or 10 nM, within an MCHreceptor ligand binding assay as described in Example 2.

A representative calcium mobilization assay is provided in Example 3.Generally preferred MCH receptor antagonists exhibit EC₅₀ values ofabout 4 micromolar or less, more preferably 1 micromolar or less, stillmore preferably about 100 nanomolar or less, 10 nanomolar or less or 1nanomolar or less within a standard in vitro MCH receptor mediatedcalcium mobilization assay, as provided in Example 3.

In certain embodiments, MCH receptor antagonists include substituted1-benzyl-4-aryl piperazine and piperidine analogues, as described withinpending U.S. patent application Ser. No. 10/152,189, filed May 21, 2002.The corresponding PCT application published as WO 02/094799 on Nov. 28,2002. This disclosure is hereby incorporated herein by reference for itsteaching of MCH receptor antagonists (pages 3-5, 20-25 and especiallyTable 1 at pages 74-107) and the preparation thereof (pages 29-42 and50-73).

Within other embodiments, MCH receptor antagonists for use within thepresent compositions are substituted benzimidazole analogues asdescribed within pending U.S. patent application Ser. No. 10/399,499,filed Jan. 9, 2003. The corresponding PCT application published as WO03/060475 on Jul. 24, 2003. This disclosure is hereby incorporatedherein by reference for its teaching of MCH receptor antagonists (pages2-5, Table I (pages 14-19) and Table II (pages 38-48)) and thepreparation thereof (pages 23-24 and 32-38).

Within further embodiments, compounds for use within the presentinvention are as described within pending U.S. patent application Ser.No. 10/399,111, filed Jan. 9, 2003. The corresponding PCT applicationpublished as WO 03/059289 on Jul. 24, 2003. This disclosure is herebyincorporated herein by reference for its teaching of MCH receptorantagonists (pages 3-4 and 31-50) and the preparation thereof (pages19-20 and 28-31).

Within further embodiments, compounds for use within the presentinvention are as described within U.S. Pat. No. 6,569,861, which ishereby incorporated by reference for its teaching ofphenylcycloalkylmethylamino and phenylalkenylamino MCH receptorantagonists (columns 3-9 and 18-19) and the preparation thereof (columns16-18).

Still further MCH receptor antagonists are described, for example,within the following published applications: WO 03/035055;US2003/0077701; WO 03/033480; WO 03/033476; WO 03/015769; WO 03/028641;WO 03/013574; WO 03/004027; WO 02/094799; WO 02/089729; WO 02/083134; WO02/076947; WO 02/076929; WO 02/057233; WO 02/051809 and WO 02/10146. Itwill be apparent that the above are illustrative examples of MCHreceptor antagonists, and are not intended to limit the scope of thepresent invention.

As noted above, compositions of the present invention may encompass apharmaceutically acceptable salt of an MCH receptor antagonist. As usedherein, a “pharmaceutically acceptable salt” is an acid or base saltthat is generally considered in the art to be suitable for use incontact with the tissues of human beings or animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication. Such salts include mineral and organic acid salts of basicresidues such as amines, as well as alkali or organic salts of acidicresidues such as carboxylic acids. Specific pharmaceutical saltsinclude, but are not limited to, salts of acids such as hydrochloric,phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic,sulfanilic, formic, toluenesulfonic, methanesulfonic, ethane disulfonic,2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic,succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH₂)_(n)—COOH where n is0-4, and the like. Similarly, pharmaceutically acceptable cationsinclude, but are not limited to sodium, potassium, calcium, aluminum,lithium and ammonium. Those of ordinary skill in the art will recognizefurther pharmaceutically acceptable salts, including those listed byRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., p. 1418 (1985). Accordingly, the present disclosure shouldbe construed to include all pharmaceutically acceptable salts of MCHreceptor antagonists.

A wide variety of synthetic procedures are available for the preparationof pharmaceutically acceptable salts. In general, a pharmaceuticallyacceptable salt can be synthesized from a parent compound that containsa basic or acidic moiety by any conventional chemical method. Briefly,such salts can be prepared by reacting the free acid or base form ofthese compounds with a stoichiometric amount of the appropriate base oracid in water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred.

Prodrugs of MCH receptor antagonists may be prepared by modifyingfunctional groups present in the compounds in such a way that themodifications are cleaved to the parent compounds. Prodrugs includecompounds wherein hydroxy, amine or sulfhydryl groups are bonded to anygroup that, when administered to a mammalian subject, cleaves to form afree hydroxyl, amino, or sulfhydryl group, respectively. Examples ofprodrugs include, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups within an MCHreceptor antagonist. Preferred prodrugs include acylated derivatives.Those of ordinary skill in the art will recognize various syntheticmethods that may be employed to prepare prodrugs of an MCH receptorantagonist.

Pharmaceutical Compositions

The practice of the present invention employs pharmaceuticalcompositions comprising an MCH receptor antagonist, together with atleast one physiologically acceptable carrier or excipient.Pharmaceutical compositions may comprise, for example, water, buffers(e.g., neutral buffered saline or phosphate buffered saline), ethanol,mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, adjuvants,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione and/or preservatives. Certainpharmaceutical compositions are formulated for oral delivery to humansor other animals (e.g., companion animals such as dogs).

If desired, other active ingredients may also be included, such asleptin, a leptin receptor agonist, a melanocortin receptor 4 (MC4)agonist, sibutramine, dexenfluramine, a growth hormone secretagogue, abeta-3 agonist, a 5HT-2 agonist, an orexin antagonist, a neuropeptide Y₁or Y₅ antagonist, a galanin antagonist, a CCK agonist, a GLP-1 agonistand/or a corticotropin-releasing hormone agonist.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal, rectalor parenteral administration. The term parenteral as used hereinincludes subcutaneous, intradermal, intravascular (e.g., intravenous),intramuscular, spinal, intracranial, intrathecal and intraperitonealinjection, as well as any similar injection or infusion technique. Incertain embodiments, compositions in a form suitable for oral use arepreferred. Such forms include, for example, tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsion,hard or soft capsules, or syrups or elixirs. Within yet otherembodiments, compositions of the present invention may be formulated asa lyophilizate.

Compositions intended for oral use may further comprise one or morecomponents such as sweetening agents, flavoring agents, coloring agentsand preserving agents in order to provide appealing and palatablepreparations. Tablets contain the active ingredient in admixture withphysiologically acceptable excipients that are suitable for themanufacture of tablets. Such excipients include, for example, inertdiluents (e.g., calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate), granulating and disintegrating agents(e.g., corn starch or alginic acid), binding agents (e.g., starch,gelatin or acacia) and lubricating agents (e.g., magnesium stearate,stearic acid or talc). The tablets may be uncoated or they may be coatedby known techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent(e.g., calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium (e.g., peanut oil, liquid paraffin or olive oil).

Aqueous suspensions comprise the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents (e.g., sodium carboxymethylcellulose,methylcellulose, hydropropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia); and dispersing orwetting agents (e.g., naturally-occurring phosphatides such as lecithin,condensation products of an alkylene oxide with fatty acids such aspolyoxyethylene stearate, condensation products of ethylene oxide withlong chain aliphatic alcohols such as heptadecaethyleneoxycetanol,condensation products of ethylene oxide with partial esters derived fromfatty acids and a hexitol such as polyoxyethylene sorbitol monooleate,or condensation products of ethylene oxide with partial esters derivedfrom fatty acids and hexitol anhydrides such as polyethylene sorbitanmonooleate). Aqueous suspensions may also contain one or morepreservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconutoil) or in a mineral oil such as liquid paraffin. The oily suspensionsmay contain a thickening agent such as beeswax, hard paraffin or cetylalcohol. Sweetening agents such as those set forth above, and/orflavoring agents may be added to provide palatable oral preparations.Such suspension may be preserved by the addition of an anti-oxidant suchas ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil (e.g., olive oil orarachis oil) or a mineral oil (e.g., liquid paraffin) or mixturesthereof. Suitable emulsifying agents may be naturally-occurring gums(e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides(e.g., soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol), anhydrides (e.g., sorbitan monoleate) andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).The emulsions may also contain sweetening and/or flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso comprise one or more demulcents, preservatives, flavoring agentsand/or coloring agents.

A pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension. The MCH receptor antagonist, dependingon the vehicle and concentration used, can either be suspended ordissolved in the vehicle. Such a composition may be formulated accordingto the known art using suitable dispersing, wetting agents and/orsuspending agents such as those mentioned above. Among the acceptablevehicles and solvents that may be employed are water, 1,3-butanediol,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils may be employed as a solvent or suspending medium.For this purpose any bland fixed oil may be employed, includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectible compositions, andadjuvants such as local anesthetics, preservatives and/or bufferingagents can be dissolved in the vehicle.

Compositions may also be prepared in the form of suppositories (e.g.,for rectal administration). Such compositions can be prepared by mixingthe drug with a suitable non-irritating excipient that is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Suitable excipientsinclude, for example, cocoa butter and polyethylene glycols.

For administration to non-human animals, the composition may also beadded to animal feed or drinking water. It may be convenient toformulate animal feed and drinking water compositions so that the animaltakes in an appropriate quantity of the composition along with its diet.It may also be convenient to present the composition as a premix foraddition to feed or drinking water.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of MCH receptor antagonist following administration). Suchformulations may generally be prepared using well known technology andadministered by, for example, oral, rectal or subcutaneous implantation,or by implantation at the desired target site. Carriers for use withinsuch formulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level of MCHreceptor antagonist release. The amount of antagonist contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

MCH receptor antagonists are generally present within a pharmaceuticalcomposition in a therapeutically effective amount. A therapeuticallyeffective amount is an amount that results in a discernible benefit in apatient carrying an MC4R mutation. Such benefit(s) include one or moreof decreased BMI, decreased food intake and/or weight loss, followingrepeated administration (e.g., from 1 to 4 times per day for a period ofweeks or months). A preferred concentration is one sufficient to inhibitthe binding of MCH to MCHR1 receptor in vitro. Compositions providingdosage levels ranging from about 0.1 mg to about 140 mg per kilogram ofbody weight per day are preferred (about 0.5 mg to about 7 g per humanpatient per day). The amount of active ingredient that may be combinedwith the carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. Dosage unit forms will generally contain from about 1 mgto about 500 mg of an active ingredient. It will be understood, however,that the optimal dose for any particular patient will depend upon avariety of factors, including the activity of the specific compoundemployed; the age, body weight, general health, sex and diet of thepatient; the time and route of administration; the rate of excretion;any simultaneous treatment, such as a drug combination; and the type andseverity of the particular disease undergoing treatment. Optimal dosagesmay be established using routine testing, and procedures that are wellknown in the art.

Methods of Use

The present invention provides methods for preventing treating healthconditions associated with MC4R mutations, such as obesity andovereating, and for reducing the body mass index of a patient carryingat least one MC4R mutation. Obesity and overeating may be diagnosed andmonitored using criteria that have been established in the art. Patientsmay include humans, domesticated companion animals (pets, such as dogs)and livestock animals, and are typically obese at the time of initiatingtreatment.

In general, prior to treatment, a determination is made as to whether ornot the patient carries at least one MC4R mutation, as defined above.Such mutations include, but are not limited to, deletion of CTCT atcodon 211, insertion of four nucleotides at codon 244, nonsense mutationat position 35, and missense mutations (e.g., Thr11Ser, Arg18Cys,Ser30Phe, Asp37Val, Val50Met, SerS8Cys, Pro78Leu, Gly98Arg, Ile102Ser,Val103Ile, Thr112Met, Ile137Thr, Thr150Ile, Arg165Trp, Ile170Val,Leu250Gln, Gly252Ser, Asn274Ser, Ile301Thr and/or Ue317Thr).

A determination as to whether or not the patient carries at least oneMC4R mutation may be made by review of the patient's chart, or usingstandard diagnostic methods. As an initial screen, a patient may (butneed not) be evaluated for characteristics commonly associated with MC4Rmutations, including early onset obesity associated with hyperphagia,tall stature, high blood pressure, hyperinsulinemia in the absence ofdiabetes and preserved reproductive function. The presence of an MC4Rmutation may be determined, for example, via PCR assay, in which an MC4Rnucleotide sequence in a sample (e.g., tissue or body fluid) obtainedfrom a patient is compared to a reference MC4R sequence for thepatient's species. Suitable PCR assays will be apparent to those ofordinary skill in the art and include, for example, assays described byHinney et al. (1999) J. Clin. Endocrinology and Metabolism 84:1483-86and Vaisse et al. (2000) J. Clin. Invest 106:253-62. It will be apparentthat this determination does not require a comparison of the completeMC4R sequences. Rather, the determination may be made by simply assayingthe patient's MC4R nucleotide sequence(s) for the presence of a specificnucleotide or series of nucleotides that are associated with obesity.

Patients who carry at least one MC4R mutations may be obese or nonobese(i.e., have never been obese or were previously obese). In either case,therapy involves administering a non-toxic melanin concentrating hormone(MCH) receptor antagonist to the patient, with dosages generally asdescribed above. For an obese patient, the amount administered isgenerally an amount that is effective to reduce (1) food consumptionand/or (2) body mass index of the patient upon sustained administration.In other words, the amount in one dose need not have a detectable effecton body mass index; however, when administered repeatedly as describedherein, the amount should be sufficient to detectably reduce foodconsumption and/or body mass index. For patients who are not currentlyobese, methods provided herein generally prevent obesity (i.e., therapyresults in a decrease in the amount of weight gained, a delay in theonset of weight gain or a maintenance of the patient's current weight).An effective amount is generally an amount that is found in clinicaltrials to decrease overeating and/or to prevent, decrease or delay theonset of weight gain in patients that carry one or more MC4R mutations.

Frequency of dosage may vary depending on the compound used and theparticular condition to be treated. In general, a dosage regimen of 4times daily or less is preferred, with 1 or 2 times daily particularlypreferred. The specific dose for any particular patient will depend upona variety of factors discussed above. In general, the use of the minimumdosage that is sufficient to provide effective therapy is preferred.Patients may generally be monitored for therapeutic effectiveness usingassays suitable for the condition being treated or prevented, which willbe familiar to those of ordinary skill in the art. For example,treatment is considered to be effective if it results in a statisticallysignificant decrease in weight, BMI or food intake.

The following Examples are offered by way of illustration and not by wayof limitation. Unless otherwise specified all reagents and solvents areof standard commercial grade and are used without further purification.

EXAMPLES Example 1 Effect of MCH Receptor Antagonist on Food ConsumptionStimulated by Reduced MC4 Receptor Activity

This Example illustrates an in vivo assay for use in confirming theability of a MCH receptor antagonist to inhibit excess food consumptionresulting from decreased MC4 activity.

Experimentally naïve male Sprague Dawley rats (Sasco, St. Louis, Mo.)weighing between 250 and 300 grams are housed in stainless steel hangingcages in a temperature and humidity controlled animal facility (22±2°C., 40-70% relative humidity) with a 12 hour light/dark cycle. Rats areimplanted with a 26 g stainless steel cannula aimed at the lateralventricle. After one week of recovery, 5, 10 or 20 mg/kg MCH receptorantagonist is administered orally in 2% d-α-tocopherol polyethyleneglycol succinate to test animals (with vehicle alone administered tocontrol animals) 30 minutes before ICV administration of 6 nmol HS014(Phoenix Peptide (Belmont, Calif.); dissolved in distilled water) ordistilled water vehicle in a volume of 5 μL. Rats are then placed intheir home cages and allowed free access to pre-weighed Purina chowpellets and water. Food consumption is measured 2 hours post ICVinjection, and the results are shown in FIG. 1.

A one-way ANOVA is conducted on the food consumption measurements.Significant dose effects (p<0.05) are further analyzed using a FisherLSD test. Animals that receive HS014 (a cyclic analogue of MSH thatfunctions as a selective MC4 receptor antagonist) eat significantly morefood than animals that receive an ICV injection of water vehicle(p<0.05). Animals administered HS014 and MCH receptor antagonist eatsignificantly less than animals that receive HS014 alone (p<0.05).Preferably, the level of food consumption in animals treated with HS014and 20 mg/kg MCH receptor antagonist is not significantly different fromthe level of consumption in animals treated with vehicle alone (i.e.,without HS014).

Example 2 Melanin Concentrating Hormone Receptor Binding Assay

This Example illustrates a standard assay of melanin concentratinghormone receptor binding that may be used to determine the bindingaffinity of compounds for the MCH receptor.

MCH1-containing membranes are prepared as described at pages 48-49 of WO03/060475. Competition binding assays are performed at room temperaturein Falcon 96 well round bottom polypropylene plates. Each assay wellcontains 150 μl of MCH1-containing membranes prepared as describedabove, 50 μl ¹²⁵I-Tyr MCH, 50 μl binding buffer, and 2 μl test compoundin DMSO. ¹²⁵I-Tyr MCH (specific activity=2200 Ci/mMol) is purchased fromNEN, Boston, Mass. (Cat # NEX 373) and is diluted in binding buffer toprovide a final assay concentration of 30 pM.

Non-specific binding is defined as the binding measured in the presenceof 1 μM unlabeled MCH. MCH is purchased from BACHEM U.S.A., King ofPrussia, Pa. (cat # H-1482). Assay wells used to determine MCH bindingcontained 150 μl of MCH receptor containing membranes, 50 μl ¹²⁵I-TyrMCH, 25 μl binding buffer, and 25 μl binding buffer.

Assay plates are incubated for 1 hour at room temperature. Membranes areharvested onto WALLAC™ glass fiber filters (PERKIN-ELMER, Gaithersburg,Md.) which are pre-soaked with 1.0% PEI (polyethyleneimine) for 2 hoursprior to use. Filters are allowed to dry overnight, and then counted ina WALLAC 1205 BETA PLATE counter after addition of WALLAC BETA SCINT™scintillation fluid.

For saturation binding, the concentration of ¹²⁵I-Tyr MCH is varied from7 to 1,000 pM. Typically, 11 concentration points are collected persaturation binding curve. Equilibrium binding parameters are determinedby fitting the allosteric Hill equation to the measured values with theaid of the computer program FitP™ (BIOSOFT, Ferguson, Mo.). For thecompounds described herein, K_(i) values are below 1 micromolar,preferably below 500 nanomolar, more preferably below 100 nanomolar.

Example 3 Calcium Mobilization Assay

This Example illustrates a representative functional assay formonitoring the response of cells expressing melanin concentratinghormone receptors to melanin concentrating hormone. This assay can alsobe used to determine if test compounds act as agonists or antagonists ofmelanin concentrating hormone receptors.

Chinese Hamster Ovary (CHO) cells (American Type Culture Collection;Manassas, Va.) are stably transfected with an MCH receptor expressionvector as described at page 50 of WO 03/060475, and are grown to adensity of 15,000 cells/well in FALCON™ black-walled, clear-bottomed96-well plates (#3904, BECTON-DICKINSON, Franklin Lakes, N.J.) in Ham'sF12 culture medium (MEDIATECH, Herndon, Va.) supplemented with 10% fetalbovine serum, 25 mM HEPES and 500 μg/mL (active) G418. Prior to runningthe assay, the culture medium is emptied from the 96 well plates. Fluo-3calcium sensitive dye (Molecular Probes, Eugene, Oreg.) is added to eachwell (dye solution: 1 mg FLUO-3 AM, 440 μL DMSO and 440 μl 20% pluronicacid in DMSO, diluted 1:4, 50 μl diluted solution per well). Plates arecovered with aluminum foil and incubated at 37° C. for 1-2 hours. Afterthe incubation, the dye is emptied from the plates, cells are washedonce in 100 μl KRH buffer (0.05 mM KCl, 0.115 M NaCl, 9.6 mM NaH₂PO₄,0.01 mM MgSO₄, 25 mM HEPES, pH 7.4) to remove excess dye; after washing,80 μl KRH buffer is added to each well. Fluorescence response ismonitored upon the addition of either human MCH receptor or testcompound by a FLIPR™ plate reader (Molecular Devices, Sunnyvale, Calif.)by excitation at 480 nM and emission at 530 nM.

In order to measure the ability of a test compound to antagonize theresponse of cells expressing MCH receptors to MCH, the EC₅₀ of MCH isfirst determined. An additional 20 μl of KRH buffer and 1 μl DMSO isadded to each well of cells, prepared as described above. 100 μl humanMCH in KRH buffer is automatically transferred by the FLIPR instrumentto each well. An 8-point concentration response curve, with final MCHconcentrations of 1 nM to 3 μM, is used to determine MCH EC₅₀.

Test compounds are dissolved in DMSO, diluted in 20 μl KRH buffer, andadded to cells prepared as described above. The 96 well platescontaining prepared cells and test compounds are incubated in the dark,at room temperature for 0.5-6 hours. It is important that the incubationnot continue beyond 6 hours. Just prior to determining the fluorescenceresponse, 100 μl human MCH diluted in KRH buffer to 2×EC₅₀ isautomatically added by the FLIPR instrument to each well of the 96 wellplate for a final sample volume of 200 μl and a final MCH concentrationof EC₅₀. The final concentration of test compounds in the assay wells isbetween 1 μM and 5 μM. Typically, cells exposed to one EC₅₀ of MCHexhibit a fluorescence response of about 10,000 Relative FluorescenceUnits. Antagonists of the MCH receptor exhibit a response that issignificantly less than that of the control cells to the p≦0.05 level,as measured using a parametric test of statistical significance.Typically, antagonists of the MCH receptor decrease the fluorescenceresponse by about 20%, preferably by about 50%, and most preferably byat least 80% as compared to matched controls.

The ability of a compound to act as an agonist of the MCH receptor isdetermined by measuring the fluorescence response of cells expressingMCH receptors, using the methods described above, in the absence of MCH.Compounds that cause cells to exhibit fluorescence above background areMCH receptor agonists. Compounds that induce no detectable increase inthe basal activity of the MCH receptor have no detectable agonistactivity and are preferred.

Example 4 MDCK Cytotoxicity Assay

This Example illustrates the evaluation of compound toxicity using aMadin Darby canine kidney (MDCK) cell cytoxicity assay.

1 μL of test compound is added to each well of a clear bottom 96-wellplate (PACKARD, Meriden, Conn.) to give final concentration of compoundin the assay of 10 micromolar, 100 micromolar or 200 micromolar. Solventwithout test compound is added to control wells.

MDCK cells, ATCC no. CCL-34 (American Type Culture Collection, Manassas,Va.), are maintained in sterile conditions following the instructions inthe ATCC production information sheet. Confluent MDCK cells aretrypsinized, harvested, and diluted to a concentration of 0.1×10⁶cells/ml with warm (37° C.) medium (VITACELL Minimum Essential MediumEagle, ATCC catalog # 30-2003). 100 μL of diluted cells is added to eachwell, except for five standard curve control wells that contain 100 μLof warm medium without cells. The plate is then incubated at 37° C.under 95% O₂, 5% CO₂ for 2 hours with constant shaking. Afterincubation, 50 μL of mammalian cell lysis solution is added per well,the wells are covered with PACKARD TOPSEAL stickers, and plates areshaken at approximately 700 rpm on a suitable shaker for 2 minutes.

Compounds causing toxicity will decrease ATP production, relative tountreated cells. The PACKARD, (Meriden, Conn.) ATP-LITE-M LuminescentATP detection kit, product no. 6016941, is generally used according tothe manufacturer's instructions to measure ATP production in treated anduntreated MDCK cells. PACKARD ATP LITE-M reagents are allowed toequilibrate to room temperature. Once equilibrated, the lyophilizedsubstrate solution is reconstituted in 5.5 mL of substrate buffersolution (from kit). Lyophilized ATP standard solution is reconstitutedin deionized water to give a 10 mM stock. For the five control wells, 10μL of serially diluted PACKARD standard is added to each of the standardcurve control wells to yield a final concentration in each subsequentwell of 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM. PACKARD substratesolution (50 μL) is added to all wells, which are then covered, and theplates are shaken at approximately 700 rpm on a suitable shaker for 2minutes. A white PACKARD sticker is attached to the bottom of each plateand samples are dark adapted by wrapping plates in foil and placing inthe dark for 10 minutes. Luminescence is then measured at 22° C. using aluminescence counter (e.g., PACKARD TOPCOUNT Microplate Scintillationand Luminescence Counter or TECAN SPECTRAFLUOR PLUS), and ATP levelscalculated from the standard curve. ATP levels in cells treated withtest compound(s) are compared to the levels determined for untreatedcells. Cells treated with 10 μM of a preferred test compound exhibit ATPlevels that are at least 80%, preferably at least 90%, of the untreatedcells. When a 100 μM concentration of the test compound is used, cellstreated with preferred test compounds exhibit ATP levels that are atleast 50%, preferably at least 80%, of the ATP levels detected inuntreated cells.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A method for treating obesity in a mammalian patient, comprisingdetermining whether or not the patient carries at least one melanocortin4 (MC4) receptor mutation that is associated with obesity and, if thepatient carries such a mutation, administering an amount of a non-toxicmelanin concentrating hormone (MCH) receptor antagonist effective toreduce either or both of (1) food consumption or (2) body mass index ofthe patient upon sustained administration.
 2. A method according toclaim 1, wherein the MCH receptor antagonist has a molecular mass lessthan 700 a.m.u. and is nonpeptidic.
 3. A method according to claim 1,wherein the MCH receptor antagonist has no detectable MCH receptoragonist activity.
 4. A method according to claim 1, wherein the MCHreceptor antagonist binds to an MCH receptor with a K_(i) that is lessthan 1 micromolar.
 5. A method according to claim 1, wherein the MCHreceptor antagonist binds to an MCH receptor with a K_(i) that is lessthan 100 nanomolar.
 6. A method according to claim 1, wherein the MCHreceptor antagonist is administered orally.
 7. A method according toclaim 1, wherein the MCH receptor antagonist is administered byinjection.
 8. A method according to claim 1, wherein the determinationof whether or not the patient carries an MC4 receptor mutation isperformed via PCR using a sample of a tissue or body fluid obtained fromthe patient.
 9. A method for treating obesity in a patient carrying atleast one MC4 receptor mutation that is associated with obesity,comprising administering an effective amount of a non-toxic MCH receptorantagonist to a patient previously determined to carry such a mutation.10. A method according to claim 9, wherein the MCH receptor antagonisthas a molecular mass less than 700 a.m.u. and is nonpeptidic.
 11. Amethod according to claim 9, wherein the MCH receptor antagonist has nodetectable MCH receptor agonist activity.
 12. A method according toclaim 9, wherein the MCH receptor antagonist binds to an MCH receptorwith a K_(i) that is less than 1 micromolar.
 13. A method according toclaim 9, wherein the MCH receptor antagonist binds to an MCH receptorwith a K_(i) that is less than 100 nanomolar.
 14. A method according toclaim 9, wherein the MCH receptor antagonist is administered orally. 15.A method according to claim 9, wherein the MCH receptor antagonist isadministered by injection.
 16. A method for preventing obesity in amammalian patient, comprising determining whether or not the patientcarries at least one MC4 receptor mutation that is associated withobesity and, if the patient carries such a mutation, administering aneffective amount of a non-toxic melanin concentrating hormone (MCH)receptor antagonist, and thereby preventing obesity in the patient. 17.A method according to claim 16, wherein the MCH receptor antagonist hasa molecular mass less than 700 a.m.u. and is nonpeptidic.
 18. A methodaccording to claim 16, wherein the MCH receptor antagonist has nodetectable MCH receptor agonist activity.
 19. A method according toclaim 16, wherein the MCH receptor antagonist binds to an MCH receptorwith a K_(i) that is less than 1 micromolar.
 20. A method according toclaim 16, wherein the MCH receptor antagonist binds to an MCH receptorwith a K_(i) that is less than 100 nanomolar.
 21. A method according toclaim 16, wherein the MCH receptor antagonist is administered orally.22. A method according to claim 16, wherein the MCH receptor antagonistis administered by injection.
 23. A method according to claim 16,wherein the determination of whether or not the patient carries an MC4receptor mutation is performed via PCR using a sample of a tissue orbody fluid obtained from the patient.
 24. (canceled)